Microfluidic device

ABSTRACT

The object of the present invention is to provide a production process of a microfluidic device in which a porous resin layer, which is capable of optimally fixing a large amount of enzyme, antigen or other protein or catalyst on the inner surface of a minute channel of a microfluidic device without obstructing said channel, is formed at a uniform thickness on the surface of said channel. 
     In the present invention, by preliminarily forming a porous resin layer having a large number of pores in its surface on a substrate, and forming an indentation having a porous resin layer on its bottom surface by using an activating energy beam-curable composition on said porous resin layer, followed by forming a channel by adhering a member serving as a cover to said indentation, a porous resin layer can easily be formed at a uniform thickness on the surface of said channel.

REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/JP04/03096 filed Mar. 10, 2004.

TECHNICAL FIELD

The present invention relates to a microfluidic device having a porousresin layer employing a three-dimensional mesh structure on the innersurface of a channel, and a production process thereof.

BACKGROUND ART

Attempts have begun in recent years to use microfluidic devices toanalyze the components of fluids containing trace amounts of DNA,biological substances and so forth in various fields including medicaldiagnostics and biochemical testing.

Microfluidic devices are also referred to as microfluid devices,microfabricated devices, lab-on-a-chip and micro total analyticalsystems (μ-TAS), and are capable of accelerating reactions and analyses,reducing the amounts of required reagents and reducing waste products bycarrying out reactions and analyses in minute, capillary channelscontained within the device.

In the case of using such microfluidic devices to react with a sample ina fluid by fixing an enzyme, catalyst or functional group and so forthon the inside surface of a channel, or in the case of detecting DNA andso forth in a sample by fixing a probe such as a DNA fragment of aspecific sequence, it is important to fix larger amounts of enzyme,catalyst or DNA fragments or other probes in order to improve reactionrate and analysis sensitivity.

The inside of the channel is preferably made to be porous in order toincrease the fixed amount of a functional group, (bio)chemical substanceor biological substance as described above in a channel. A known exampleof a channel in which a porous body is formed inside is that in whichthe entire inside of the channel is a porous body, and is formed by amethod in which a silicon or aluminum sheet is made to be porous byetching and heat treatment within a range to a fixed depth from thesurface of the sheet, followed by adhering a cover onto said poroussurface (see Japanese Unexamined Patent Application, First PublicationNo. H6-169756). However, since the entire inside of this channel isporous, and the fluid that flows through this channel flows through thepores of said porous body, a high pressure of several hundred Kpa isrequired for the fluid to flow at an adequate flow rate through thechannel. Consequently, the microfluidic device body and connecting portsfor introducing the fluid were required to be of a rugged structurecapable of withstanding high pressure. In addition, since the fixationdensity of these inorganic materials is small in addition to the typesof functional groups introduced onto the surface being limited, theystill ended up being inadequate even if a porous body was employed.Moreover, since silicon and metal are optically opaque, therebypreventing pigments and fluorescent pigments fixed to the inside of theporous body from being observed from the outside, they did notcontribute to improvement of the sensitivity of light absorption orfluorescent detection. Since these materials are optically opaque, thereis a large amount of scattering of excitation light during fluorescencemeasurement, and since excitation light unable to be completely cut outwith a filter ends up entering the receiving side, the baseline ofmeasured fluorescent intensity becomes higher, thereby inviting adecrease in the S/N ratio and a decrease in reliability. Moreover, sincesilicon and metal have high thermal conductivity, it is difficult toprovide a temperature gradient in a channel, thereby placing limitationson use as a microfluidic device.

On the other hand, since resins (organic polymers) have numerous typesof functional groups that can be introduced onto their surface, and thefixation densities of those functional groups are high, they arepreferable for use as constituent materials of the inner surface of thechannels of microfluidic devices (see Japanese Unexamined PatentApplication, First Publication No. 2000-2705). However, in processesinvolving the formation of a grooved channel like that known as aproduction process of a resin microfluidic device (see, for example,Japanese Unexamined Patent Application, First Publication No.2000-46797), although a process is described in which a grooved channelhaving minute surface irregularities on the bottom is formed by formingminute surface irregularities on the surface of a base material byelectron etching, coating an activating energy beam-curable compoundthereon, radiating an energy beam onto those portions other than thechannel to cure the activating energy beam-curable compound, andremoving the uncured activating energy beam-curable compound of thenon-irradiated portion, since this only involves the providing ofsurface irregularities for imparting hydrophilicity to the channelbottom of said microfluidic device, a three-dimensional mesh-like porouslayer is not formed. Although it is not known whether or not more probeis fixed by this microfluidic device having an inner surface withsurface irregularities than by a microfluidic device in which the innersurface is not treated, according to a confirmatory experiment conductedby the inventors of the present invention, the degree of the increasewas small and was not considered to be adequate.

On the other hand, a method of increasing the fixed amount of an enzymeor catalyst on the surface of a sheet and so forth instead of thesurface of a channel is disclosed in which a thin porous layer is formedon the surface of said sheet followed by fixing the enzyme or catalystthereon (see Japanese Unexamined Patent Application, First PublicationNo. 2000-2705). However, a method in which such a porous layer isprovided on one side of the inner surface of a minute channel of amicrofluidic device has heretofore not been known.

In addition, a method of producing a hydrophilic porous membrane isdisclosed in which a mixed solution of an energy beam-curable resin,linear polymer and solvent is coated onto a base material and irradiatedwith energy followed by contacting with a non-solvent of the linearpolymer to cause phase separation (see Japanese Unexamined PatentApplication, First Publication No. 10-007,835). However, with respect tothis method as well, a method of providing such a resin at a uniformthickness on the inner surface of a minute channel of a microfluidicdevice has heretofore not been known.

The objects to be solved by the present invention consist of providing amicrofluidic device, in which a porous resin layer having athree-dimensional mesh structure, which is capable of optimally fixing alarge amount of enzyme, antigen or other protein or catalyst on theinner surface of a minute channel of a microfluidic device withoutobstructing the channel of the microfluidic device, is formed at auniform thickness on the surface of said channel, a microfluidic devicein which said porous resin layer is formed at an arbitrary location inthe direction of flow of the channel, a microfluidic device in whichsaid porous resin layer is formed on a portion of the cross-section ofthe channel, and a production process of said microfluidic device.

DISCLOSURE OF THE INVENTION

As a result of conducting extensive studies on ways of achieving theaforementioned objects, the inventors of the present invention foundthat the aforementioned objects can be achieved by preliminarily forminga porous resin layer having a three-dimensional mesh structure on thesurface of a substrate, and forming an indentation having a porous resinlayer having a three-dimensional mesh structure on its bottom surface insaid porous resin layer by using an activating energy beam-curablecomposition, followed by forming a channel by adhering a member servingas a cover to said indentation, thereby leading to completion of thepresent invention.

Namely, the present invention provides a microfluidic device comprisinga substrate, a porous resin layer having a three-dimensional meshstructure, a channel and a cover; wherein, said microfluidic device (I)has said porous resin layer in the upper portion of the substrate, (II)said porous resin layer is filled with a curable resin of an activatingenergy beam-curable resin composition (X) impregnated excluding thechannel portion, and (III) the channel has wall surfaces consisting of aporous resin layer having a three-dimensional mesh structure that is notfilled with activating energy beam-curable resin composition (X), acurable resin layer of the activating energy beam-curable resincomposition (X) formed in the upper portion of the porous resin layerhaving a three-dimensional mesh structure filled with the curable resinof activating energy beam-curable resin composition (X), and a cover,and is formed into the form of a cavity.

In addition, the present invention provides a production process of amicrofluidic device comprising: (1) a step in which a porous resin layerhaving a three-dimensional mesh structure having a large number of poresis formed on the surface of a substrate; (2) a step in which anactivating energy beam-curable composition (X) containing an activatingenergy beam-polymerizeable compound (a) is coated onto said porous resinlayer, an uncured coating of said composition (X) is formed, anactivating energy beam is radiated on the uncured coating other than atthe portion to serve as the channel to form a cured or semi-curedcoating of the compound (X), the uncured compound (X) at thenon-irradiated portion is removed, and an indentation is formed in whicha porous resin layer having a three-dimensional mesh structure isexposed on the bottom surface; and (3) another member serving as a coveris adhered to the indentation of the member having the indentation sothat the indentation serves as a channel in the shape of a cavity;wherein, more preferably, the step in which a porous resin layer havinga three-dimensional mesh structure is formed on the surface of thesubstrate consists of coating onto the substrate an activating energybeam-curable membrane deposition liquid (J) that contains an activatingenergy beam-polymerizeable compound (b) and a poor solvent (R) that iscompatible with said compound (b) but incompatible with a polymer formedfrom said compound (b), followed by radiating an activating energy beamonto said membrane deposition liquid (J) and forming a porous resinlayer having a three-dimensional mesh structure on the surface of thesubstrate.

In the case of using a microfluidic device of the present invention as aliquid chromatography member, since high-speed analysis is possible evenif the developer is allowed to flow at low pressure, it is not necessaryto give high pressure resistance to separation columns or connectionsbetween feed lines containing developer, and since the entire μ-TASdevice does not require a rugged structure due to the ease ofincorporating into μ-TAS, the separation target can be preferablyseparated even for minute sample volumes. In addition, since the fixedamounts of (bio) chemical substances having functional groups ormolecule-recognizing functions can be made to be extremely large ascompared with the prior art, the allowable sample feed volume can beincreased resulting in improved quantification and accuracy as well asimproved sensitivity. Moreover, since the device has high productivityand can be produced inexpensively, it can also be used for disposableapplications.

In addition, since a porous resin layer having a three-dimensional meshstructure is able to be only formed on the bottom surface of thechannel, the surface of said porous resin layer can be observedoptically through the channel, thereby allowing highly sensitive andhighly quantitative measurements.

In addition, in the case of using a microfluidic device of the presentinvention as a member for affinity electrophoresis, analyses can beperformed by using as an electrophoresis medium without using a sol orgel, thereby eliminating the need for a complex preparation procedureprior to use, reducing deterioration of performance during marketdistribution and enabling storage in a dry state to facilitate storageand market distribution. In addition, since the fixed amounts of(bio)chemical substances having functional groups andmolecule-recognizing functions can be made to be extremely large ascompared with the prior art, the allowable sample feed volume can beincreased resulting in improved quantification and accuracy as well asimproved sensitivity. Moreover, since a porous resin layer having athree-dimensional mesh structure can be formed only on the bottomsurface of the channel, the surface of said porous resin layer can beoptically observed throughout the channel, thereby enabling highlysensitive and highly quantitative measurements.

In the case of using a microfluidic device of the present invention as amicroarray member for DNA analysis or immunodiagnosis and so forth, thefixed amount of probe can be made to be extremely large as compared withthe prior art, thereby resulting in improved detection sensitivity,improved quantitativeness and faster analyses. In addition, since theporous resin layer having a three-dimensional mesh structure is formedas a spot inside the channel and a different probe can be fixed at eachspot, multiple parameters can be analyzed with a single channel.Moreover, since the porous resin layer having a three-dimensional meshstructure is able to be formed only on the bottom surface of thechannel, the surface of said porous resin layer can be opticallyobserved throughout the channel, thereby enabling highly sensitive andhighly quantitative measurements.

In the case of using a microfluidic device of the present invention as areaction tank or reaction tube of a microreactor, since the fixedamounts of catalysts, enzymes and so forth can be increased, reactionspeed and yield are improved. In addition, since the porous resin layerhaving a three-dimensional mesh structure can be formed at an arbitraryregion inside the channel, and a different catalyst and so forth can befixed at each region, multiple stages of reactions can be carried outwith a single channel.

Since the use of a production process of the present inventioneliminates the difficulty and restrictions of having to subsequentlyform a porous resin layer in a narrow channel, a microfluidic devicehaving a porous resin layer having a three-dimensional mesh structure ofuniform thickness on the surface of a channel can be easily producedwithout obstructing said channel on the inner surface of the minutechannel of the microfluidic device. In addition, the thickness, poreshape and pore diameter of said porous resin layer can be easilyadjusted to optimum values for the purpose of use. Moreover, a porousresin layer forming portion having a three-dimensional mesh structure ofan arbitrary length, such as a spot-shaped porous resin layer portion,is easily provided in a portion of the channel in the direction of flow.In addition, there is no leakage of fluid inside the channel through aportion of the porous resin layer other than the porous resin layer onthe bottom of the channel.

According to a production process of the present invention, since thepore diameter and thickness of a porous resin layer having athree-dimensional mesh structure formed on the inner surface of a minutechannel can be easily adjusted to optimum values for the purpose of use,specific surface area can be increased, a large amount of substance canbe fixed, and (bio)chemical analysis and detection can be carried out ina short period of time with high sensitivity and high accuracy using aminimum amount of reagents. In addition, since a porous resin layerhaving a three-dimensional mesh structure can be formed only on thebottom surface of the channel, the surface of said porous resin layercan be optically observed throughout the channel, thereby enablinghighly sensitive measurements in the case of using for analysis.Moreover, a porous resin layer forming portion of an arbitrary length,such as a spot-shaped porous resin layer portion, is easily provided ina portion of the channel in the direction of flow.

In addition, by providing a step in which the surface of a porous resinlayer having a three-dimensional mesh structure is surface treated ormodified following a step in which said porous resin layer is formed,reactive functional groups can be introduced onto the surface of saidporous resin layer, and various types of (bio)chemical substances andbiological substances can be fixed to the surface of said porous resinlayer by covalent bonding by reacting with these functional groups.Naturally, these substances can also be fixed by adsorption by ionicbonding or hydrophobic bonding. At this time, a plurality of porousresin layer spots can be formed as previously described, different typesand concentrations of functional groups can be introduced at each ofsaid plurality of porous spots, or different types of substances caneasily be fixed.

Moreover, as a result of making the viscosity of composition (X) 30 to3000 mPa·s at 25° C., composition (X) rapidly penetrates the porousresin layer having a three-dimensional mesh structure when composition(X) is coated onto said porous resin layer, thereby allowing amicrofluidic device to be produced easily without leakage of fluid inthe channel through a portion of the porous resin layer other than theporous resin layer on the bottom of the channel. In addition, as aresult of making the viscosity within the aforementioned range,composition (X) is completely removed from the porous resin layer whenuncured composition (X) of the non-irradiated portion is removed afterradiating with the activating energy beam.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scanning electron micrograph of a porous resin layer havinga three-dimensional mesh structure produced in Example 1.

BEST MODE FOR CARRYING OUT THE INVENTION

A production process of a microfluidic device having a three-dimensionalmesh structure, porous resin layer on the inner surface of a channel ofthe present invention is at least comprised of the following steps (1)to (3):

-   (1) a step in which a porous resin layer having a three-dimensional    mesh structure having a large number of pores is formed on the    surface of a substrate;-   (2) a step in which an activating energy beam-curable    composition (X) containing an activating energy beam-polymerizeable    compound (a) is coated onto said porous resin layer, an uncured    coating of said composition (X) is formed, an activating energy beam    is radiated onto the uncured coating other than at the portion to    serve as the channel to form a cured or semi-cured coating of the    compound (X), the uncured compound (X) at the non-irradiated portion    is removed, and an indentation is formed in which a porous resin    layer having a three-dimensional mesh structure is exposed on the    bottom surface; and-   (3) another member serving as a cover is adhered to the indentation    of the member having the indentation so that the indentation serves    as a channel in the shape of a cavity.

A method of forming a porous resin layer having a three-dimensional meshstructure having a large number of pores in step (1) may be an arbitrarymethod provided said porous resin layer can be formed, and any of thefour methods indicated below, for example, can be used. Athree-dimensional mesh structure referred to here refers to a structurein which pores (voids) and a resin that serves as their matrix arerespectively connected in the directions of three dimensions, and saidpores open onto the surface of a porous resin layer. Examples include astructure in which air bubble-like cavities are mutually connected (alsoreferred to as a sponge-like structure), a structure in which voidsbetween mutually adhered resin particles are connected to form pores(also referred to as an aggregated particle-like structure or sinteredbody-like structure), a structure that is intermediate to these twostructures in which the pores and resin have a nearly equivalentstructure, and their respective layers are mutually connected (alsoreferred to a modulated structure or gyroid structure), and a non-wovenfabric-like structure (also referred to as a matte-like structure).

A first method of forming a porous resin layer having athree-dimensional mesh structure consists of forming a porous resinlayer having a three-dimensional mesh structure by coating an activatingenergy beam-curable membrane deposition liquid (J) (to be referred to asmembrane deposition liquid (J)) containing an activating energybeam-polymerizeable compound (b) (to be referred to as polymerizeablecompound (b)) and a poor solvent (R) that is compatible with theaforementioned compound (b) but incompatible with a polymer formed fromcompound (b) to polymerize the aforementioned compound (b) and causephase separation (this method is to be referred to as the reactioninduction-type phase separation method). In this method, as a result ofpolymerization of compound (b), poor solvent (R) becomes no longercompatible with the polymer that is formed, phase separation occursbetween the polymer and poor solvent (R), and poor solvent (R) isincorporated inside and between the deposited polymer. By then removingthis poor solvent (R), the regions occupied by poor solvent (R) becomepores, thereby enabling the formation of a porous resin layer having athree-dimensional mesh structure.

Polymerizeable compound (b) is polymerized by an activating energy beamin the presence or absence of a polymerization initiator, and ispreferably an addition polymerizeable compound or compound havingpolymerizeable carbon-carbon double bonds as activated energybeam-polymerizeable functional groups, with highly reactive(meth)acrylic compounds, vinyl ethers and maleimide compounds that arecured even in the absence of a photopolymerization initiator beingparticularly preferable. Moreover, polymerizeable compound (b) is morepreferably a compound that forms a crosslinked compound when polymerizedsince it is capable of enhancing shape retention in the semi-cured stateand increasing strength after curing. Consequently, it is even morepreferably a compound that has two or more polymerizeable carbon-carbondouble bonds in a single molecule (the having of two or more additionpolymerizeable functional groups in a single molecule is hereinafterreferred to as being “multifunctional”).

Examples of compounds that can be used for such a polymerizeablecompound (b) include (meth)acrylic monomers, maleimide-based monomersand polymerizeable oligomers having a (meth)acryloyl group or maleimidegroup in their molecular chain.

Examples of the aforementioned (meth)acrylic monomers includedifunctional monomers such as diethylene glycol di(meth)acrylate,neopentyl glycol di(meth)acrylate, 1,6-hexanediol di(meth)acrylate,2,2′-bis(4-(meth)acryloyloxypolyethyleneoxyphenyl)propane,2,2′-bis(4-(meth)acryloyloxypolypropyleneoxyphenyl)propane,hydroxydipivalic acid neopentyl glycol di(meth)acrylate, dicyclopentanyldiacrylate, bis(acryloxyethyl)hydroxyethylisocyanurate andN-methylenebisacrylamide; trifunctional monomers such as trimethylolpropane tri(meth)acrylate, trimethylol ethane tri(meth)acrylate,tris(acryloxyethyl)isocyanurate and caprolactone-modifiedtris(acryloxyethyl)isocyanurate; tetrafunctional monomers such aspentaerythritol tetra(meth)acrylate; and hexafunctional monomers such asdipentaerythritol hexa(meth)acrylate.

Examples of maleimide-based monomers include difunctional monomers suchas 4,4′-methylenebis(N-phenylmaleimide),2,3-bis(2,4,5-trimethyl-3-thienyl)maleimide, 1,2-bismaleimide ethane,1,6-bismaleimide hexane, triethylene glycol bismaleimide,N,N′-m-phenylenedimaleimide, m-tolylendimaleimide,N,N′-1,4-phenylenedimaleimide, N,N′-diphenylmethane dimaleimide,N,N′-diphenyl ether dimaleimide, N,N′-diphenylsulfone dimaleimide,1,4-bis(maleimidoethyl)-1,4-diazoniabicyclo-[2,2,2]octane dichloride,and 4,4′-isopropylidenediphenyl=dicyanate·N,N′-(methylenedi-p-phenylene) dimaleimide; andmaleimides having a maleimide group such as N-(9-acridinyl)maleimide anda polymerizeable functional group other than a maleimide group. Thesemaleimide-based monomers can also be copolymerized with compounds havingpolymerizeable carbon-carbon double bonds such as vinyl monomers, vinylethers and acrylic monomers.

Examples of polymerizeable oligomers having a (meth)acryloyl group ormaleimide group in their molecular chain include those having a weightaverage molecular weight of 500 to 50000, specific examples of whichinclude (meth)acrylic acid esters of epoxy resin, (meth)acrylic acidesters of polyether resin, (meth)acrylic acid esters of polybutadieneresin, and polyurethane resins having a (meth)acryloyl group on the endof their molecule.

These examples of polymerizeable compound (b) can be used alone or as amixture of two or more types. In addition, they may also be used bymixing with monofunctional monomers such as monofunctional (meth)acrylicmonomers and monofunctional maleimide-based monomers for the purpose ofadjusting viscosity, adjusting adhesion or stickiness in the semi-curedstate, or imparting functions such as reactivity or hydrophilicity. Forexample, amphiphilic compound (c) to be described later may also beadded.

Examples of monofunctional (meth) acrylic monomers include methylmethacrylate, alkyl (meth)acrylate, isobornyl (meth)acrylate, alkoxypolyethylene glycol (meth)acrylate, phenoxy dialkyl (meth)acrylate,phenoxy polyethylene glycol (meth)acrylate, alkylphenoxy polyethyleneglycol (meth)acrylate, nonylphenoxy polypropylene glycol (meth)acrylate,hydroxyalkyl (meth)acrylate, glycerol acrylate methacrylate, butanediolmono(meth)acrylate, 2-hydroxy-3-phenoxypropyl acrylate,2-acryloyloxyethyl-2-hydroxypropylacrylate, ethylene oxide-modifiedphthalic acid acrylate, ω-carboxycaprolactone monoacrylate,2-acryloyloxy propylhydrodiene phthalate, 2-acryloyloxy ethyl succinate,acrylic acid dimer, 2-acryloyloxy propylyhexahydrohydrodiene phthalate,fluorine-substituted alkyl(meth)acrylate, chlorine-substitutedalkyl(meth)acrylate, sodium sulfonate ethoxy(meth)acrylate, sulfonicacid-2-methylpropane-2-acrylamide, phosphoric acid estergroup-containing (meth)acrylate, glycidyl(meth)acrylate, 2-isocyanatoethyl(meth)acrylate, (meth)acryloyl chloride, (meth)acrylaldehyde,sulfonic acid ester group-containing (meth)acrylate, silanogroup-containing (meth)acrylate, ((di)alkyl)amino group-containing(meth)acrylate, quaternary ((di)alkyl)ammonium group-containing(meth)acrylate, (N-alkyl)acrylamide, (N,N-dialkyl)acrylamide andacroloyl morpholine.

Examples of monofunctional maleimide-based monomers include N-alkylmaleimides such as N-methyl maleimide, N-ethyl maleimide, N-butylmaleimide and N-dodecyl maleimide; N-alicyclic maleimides such asN-cyclohexyl maleimide; N-benzyl maleimide; N-(substituted ornon-substituted phenyl) maleimides such as N-phenyl maleimide,N-(alkylphenyl) maleimide, N-dialkoxyphenyl maleimide,N-(2-chlorophenyl) maleimide and 2,3-dichloro-N-(2,6-diethylphenyl)maleimide and 2,3-dichloro-N-(2-ethyl-6-methylphenyl) maleimide;maleimides having a halogen such as N-benzyl-2,3-dichloromaleimide andN-(4′-fluorophenyl)-2,3-dichloromaleimide; maleimides having a hydroxylgroup such as hydroxyphenyl maleimide; maleimides having a carboxy groupsuch as N-(4-carboxy-3-hydroxyphenyl) maleimide; maleimides having analkoxy group such as N-methoxyphenyl maleimide; maleimides having anamino group such as N-[3-(diethylamino)propyl]maleimide; maleimideshaving a multicyclic aromatic group such as N-(1-pyrenyl) maleimide; andmaleimides having a polycyclic aromatic compound such asN-(dimethylamino-4-methyl-3-coumarinyl) maleimide andN-(4-anilino-1-naphthyl) maleimide.

Monomers having functional groups and ionic functional groups capable ofserving as anchors for fixing bio(chemical substances and biologicalsubstances, and for example, functional groups listed as examples offunctional groups that can be preferably inserted into a porous resinlayer having a three-dimensional mesh structure to be described later,are preferably used for these monofunctional monomers.

A poor solvent that is compatible with polymerizeable compound (b) butnot compatible (not mutually soluble) with a polymer produced frompolymerizeable compound (b) is used for poor solvent (R) used in thereaction induction-type phase separation method. The degree ofcompatibility between poor solvent (R) and polymerizeable compound (b)should be such that allows the obtaining of a homogeneous membranedeposition liquid (J). Poor solvent (R) may be a single solvent or amixed solvent, and in the case of a mixed solvent, its compositecomponents alone may be that which is not compatible with polymerizeablecompound (b) or that which dissolves a polymer of polymerizeablecompound (b). Examples of poor solvent (R) include alkyl esters of fattyacids such as methyl decanoate, methyl octanoate and diisobutyl adipate;ketones such as diisobutyl ketone; alcohols such as decanol; and,mixtures of alcohol and water such as a mixture of 2-propanol or ethanoland water.

In a reaction induction-type phase separation method, the pore diameterand strength of the resulting porous resin layer having athree-dimensional mesh structure vary according to the content ofcompound (b) contained in membrane deposition liquid (J). Although thestrength of said porous resin layer is improved the greater the contentof compound (b), pore diameter tends to become smaller. The content ofcompound (b) is preferably within the range of 15 to 50% by weight, andmore preferably 25 to 40% by weight. If the content of compound (b) isless than 15% by weight, the strength of said porous resin layerdecreases, while if the content of compound (b) exceeds 50% by weight,it becomes increasingly difficult to adjust the pore diameter of saidporous resin layer.

Various additives such as a polymerization initiator, solvent,surfactant, polymerization inhibitor or polymerization retardant may beadded to membrane deposition liquid (J) in order to adjust thepolymerization rate, degree of polymerization or pore diameterdistribution and so forth.

There are no particular limitations on the polymerization initiatorprovided it is active with respect to an activating energy beam andpolymerizes polymerizeable compound (b), and radical polymerizationinitiators, anionic polymerization initiators or cationic polymerizationinitiators can be used, examples of which include acetophenones such asp-tert-butyltrichloroacetophenone, 2,2′-diethoxyacetophenone and2-hydroxy-2-methyl-1-phenylpropan-1-one; ketones such as benzophenone,4,4′-bisdimethylaminobenzophenone, 2-chlorothioxantone,2-methylthioxantone, 2-ethylthioxantone and 2-isopropylthioxantone;benzoin ethers such as benzoin, benzoin methyl ether, benzoin isopropylether and benzoin isobutyl ether; benzyl ketals such as benzyl dimethylketal and hydroxycyclohexyl phenyl ketone; and, azides such asN-azidosulfonyl phenyl maleimide. In addition, polymerizeablephotopolymerization initiators such as maleimide-based compounds canalso be used.

Examples of polymerization retardants and polymerization inhibitorsinclude α-methyl styrene, 2,4-diphenyl-4-methyl-1-pentene and otheractivating energy beam-polymerizeable compounds such as vinyl monomershaving a low polymerization rate; and, hindered phenols such astert-butylphenol.

In addition, there are no particular limitations on the solvent added,and examples include alcohols such as ethanol, ketones such as acetone,amide-based solvents such as N,N-dimethylformamide and chlorine-basedsolvents such as methylene chloride.

In addition, known, commonly used surfactants, hydrophobic compounds,thickeners, modifiers, colorants, fluorochromes, ultraviolet absorbers,enzymes, proteins, cells or catalysts and so forth can also be added toimpart functions such as coatability and smoothness, or to adjustpattern resolution or the degree of hydrophilicity during lithographicpattern formation.

A substrate that can be used in a reaction induction-type phaseseparation method should be that which is substantially not attacked,for example not dissolved or decomposed, by membrane deposition liquid(J) or activating energy beam used.

Examples of such substrates include polymers; crystals such as glass orquartz; ceramics; semiconductors such as silicon; and metals, withpolymers being particularly preferable. A polymer used for the substratemay be a homopolymer or copolymer, or a thermoplastic polymer orheat-curable polymer. In addition, the substrate may be composed of apolymer blend or polymer alloy, and may be a laminate or other compositematerial. Moreover, the substrate may also contain an additive such as amodifier, colorant, filler or reinforcing agent.

If a reaction induction-type phase separation method is used, a porousresin layer having a three-dimensional mesh structure can be formed inthe manner of an aggregated particulate structure in which particulatepolymers having a diameter of about 0.1 to 1 μm are mutually aggregated,or a sponge-like structure in which air bubbles having a diameter of 0.1to 1 μm are mutually connected. In addition, in said reactioninduction-type phase separation method, although a so-called isotropicmembrane is normally formed in which the pore diameter of the pores isuniform in the direction of membrane thickness, a so-calledheterogeneous membrane (also referred to as an asymmetrical membrane),in which pore diameter is distributed in the direction of membranethickness, can also be formed by adding a volatile solvent to membranedeposition liquid (J), coating that liquid and then volatilizing andremoving a portion thereof prior to irradiation with an activatingenergy beam. At this time, a layer having a small pore diameter (alsoreferred to as a dense layer) can be formed on the surface in contactwith the substrate on which is coated membrane deposition liquid (J) byadding a volatile good solvent, while a dense layer can be formed on theopposite side of the substrate by adding a volatile poor solvent ornon-solvent. According to said reaction induction-type phase separationmethod, a porous resin layer having a three-dimensional mesh structurecan be formed having a pore diameter of, for example, 0.05 to 5 μm.

In the present reaction induction-type phase separation method, in thecase of forming a region or plurality of regions to which a porous resinlayer having a three-dimensional mesh structure is limited, said porousresin layer can be formed on a portion of a substrate by, for example,(a) a method in which an activating energy beam-curable resincomposition (X) is coated onto a portion of a substrate by a silk screenmethod and so forth followed by exposure, or (b) a method in which anactivating energy beam-curable resin composition (X) is coated onto anentire substrate followed by pattern exposure. The present first methodallows a functional group to be easily introduced into said porous resinlayer by using a compound (b) having a functional group.

A second method of forming a porous resin layer having athree-dimensional mesh structure is a method in which, after contactinga substrate and a solvent (S) capable of dissolving or swelling saidsubstrate, the solvent (S) is washed off using a solvent (T) that iscompatible with solvent (S) but does not dissolve or swell saidsubstrate to form said porous resin layer (said method is hereinafter tobe referred to as the “surface swelling method”). In said method, apolymer that is dissolved or swollen by a solvent is used for thesubstrate, a solvent is contacted with the surface of said polymer, andafter dissolving or swelling a portion of said substrate, by washingwith a solvent that is not compatible with said polymer, said polymeraggregates in the form of a mesh resulting in the formation of a porousresin layer having a three-dimensional mesh structure.

Examples of a substrate used in the surface swelling method includestyrene-based polymers such as polystyrene, poly-α-methylstyrene,polystyrene/maleic acid copolymer and polystyrene/acrylonitrilecopolymer; polysulfone-based polymers such as polysulfone and polyethersulfone; (meth)acrylic polymers such as polymethyl methacrylate andpolyacrylonitrile; polymaleimide-based polymers; polycarbonate-basedpolymers such as bisphenol A-based polycarbonate, bisphenol F-basedpolycarbonate and bisphenol Z-based polycarbonate; cellulose-basedpolymers such as cellulose acetate and methyl cellulose;polyurethane-based polymers; polyamide-based polymers; and,polyimide-based polymers.

There are no particular limitations on solvent (S) in the surfaceswelling method provided it is capable of dissolving or swelling theaforementioned substrate, examples of which include amide-based solventssuch as N,N-dimethylformamide and N,N-dimethylacetoamide,dimethylsulfoxide and chloride-based solvents such as methylenechloride. In addition, these solvents can also be used in the form ofmixed solvents by mixing.

Solvent (T) is a solvent that is miscible with solvent (S) and does notdissolve the substrate. Examples of solvent (T) include water, alcoholssuch as propanol, and mixtures of water and alcohol.

Examples of methods for contacting the substrate with solvent (S)include immersing the substrate in solvent (S), or spraying or spreadingsolvent (S) on the substrate surface.

Although examples of washing off solvent (S) with solvent (T) include amethod in which solvent (S) is washed off by immersing in solvent (T),and a method in which solvent (S) is washed off by spraying with solvent(T), a method in which the entire substrate is immersed in solvent (T)is preferable.

A porous resin layer having a three-dimensional mesh structure producedby a surface swelling method is integrated with a substrate and is ableto form a sponge-like or aggregated particle structure. The thickness ofsaid porous resin layer can be controlled according to the duration ofcontact between the substrate and solvent (S), and the shorter theduration of contact, the less the thickness of said porous resin layer.It is necessary to suitably adjust the duration of contact between thesubstrate and solvent (S) according to the material and thickness of thesubstrate used, the type of solvent and so forth. If the duration ofcontact is too short, dissolution of the substrate does not proceedadequately and pores are not adequately formed. In addition, if theduration of contact is too long, the strength of the substratedecreases.

In the present surface swelling method, in the case of forming a porousresin layer having a three-dimensional mesh structure in a limitedregion or plurality of regions, a porous resin layer having athree-dimensional mesh structure can be formed on a portion of thesubstrate by using, for example, a method in which portions other thanthat where said porous resin layer is to be formed are covered withmasking tape.

A third method of forming a porous resin layer having athree-dimensional mesh structure consists of forming a porous resinlayer having a three-dimensional mesh structure on the surface of asubstrate by coating a membrane deposition liquid (K), comprised bydissolving a linear polymer (P) in a solvent (U), and aggregating thelinear polymer (P) in a porous form by contacting said substrate with asolvent (V) that does not dissolve or cause swelling of said linearpolymer (P) and is compatible with solvent (U) (to be referred to as thewet method).

A linear polymer that forms a porous resin layer having athree-dimensional mesh structure by dissolving in solvent (U) can beused for a linear polymer (P) that can be used in the wet method. Linearpolymers (P) such as styrene-based polymers, sulfone-based polymers,vinyl-based polymers, amide-based polymers, imide-based polymers,cellulose-based polymers, polycarbonates, acrylic polymers and so forthare preferable since they allow costs to be lowered and are handledeasily.

A solvent similar to solvent (S) that can be used in the aforementionedsurface swelling method can be used for solvent (U) in the wet method,while a solvent similar to solvent (T) in the aforementioned surfaceswelling method can be used for solvent (V).

In addition, various types of additives such as additives able to beused in the previously described reaction induction-type phaseseparation method may be added to membrane deposition liquid (K) asnecessary.

Any arbitrary substrate may be used for the substrate used in the wetmethod, and although a substrate that is substantially attacked bymembrane deposition liquid (K), in which linear polymer (P) is dissolvedin solvent (U), is preferable, if a substrate is used that dissolves orswells in the aforementioned membrane deposition liquid (K), a porousresin layer having a three-dimensional mesh structure can be formed by amechanism in which the aforementioned surface swelling method is addedto the present wet method. Examples of such substrates include polymers;crystals such as glass and quartz; ceramics; semiconductors such assilicon; and metals. Polymers are particularly preferable.

The porous structure obtained according to the wet method may be asponge-like structure, aggregated particle-like structure, gyroidstructure or other complex shape having macrovoids.

In the case of using the wet method, although a heterogeneous membrane(asymmetrical membrane) having a dense layer is normally formed on theopposite side of a coated substrate, an isotropic membrane can also beformed by adding a salt or other low molecular weight compound (poreforming agent) or adjusting the boiling point of the poor solvent orgood solvent and so forth. In addition, a porous resin layer having athree-dimensional mesh structure and pore diameter of 0.005 to 2 μm canbe formed by adjusting the concentration of linear polymer (P), amountof solvent added and so forth.

In the present wet method, in the case of forming a porous resin layerhaving a three-dimensional mesh structure in a limited region orplurality of regions, a porous resin layer having a three-dimensionalmesh structure can be formed on a portion of a substrate by, forexample, a method in which membrane deposition liquid (K) is only coatedat the portion where said porous resin layer is formed by screenprinting and so forth, or a method in which those portions other thanwhere said porous resin layer is formed are covered with masking tape.

A fourth method of forming a porous resin layer having athree-dimensional mesh structure consists of a method in which a porousresin layer having a three-dimensional mesh structure is formed bycoating a membrane deposition liquid (L), in which an activating energybeam-polymerizeable compound (d), linear polymer (Q) and solvent (W)that dissolves them both are uniformly mixed, onto a substrate,polymerizing the polymerizeable compound (d) in solution by irradiatingwith an activating energy beam, and aggregating the linear polymer in aporous form by contacting said substrate with a solvent (N) that doesnot dissolve said linear polymer (Q) and is compatible with solvent (W)(to be referred to as energy beam-wet method).

In the present method, by using an activating energy beam-polymerizeablecompound having a hydroxyl group, amino group, carboxyl group, aldehydegroup, epoxy group or other arbitrary functional group for activatingenergy beam-polymerizeable compound (d) in the same manner as describedin the section on activating energy beam-polymerizeable compound (b),these functional groups can be introduced onto the surface of a porousresin layer having a three-dimensional mesh structure more efficientlythan the aforementioned reaction induction-type phase separation method.

Activating energy beam-polymerizeable compound (d) is preferably acrosslinking polymerizeable compound, and can be used by suitablyselecting from the compounds listed as examples of the aforementionedactivating energy beam-polymerizeable compound (b). Although the curedproduct of activating energy beam-polymerizeable compound (d) may besoluble or insoluble in solvent (W), in order to efficiently arrange thefunctional groups possessed by said compound (d) on the pore surface,said cured product is preferably soluble in solvent (W). Furthermore,when the cured product of said compound (d) is a crosslinked polymer,the term “soluble” should be read as “gelled”, while the term“insoluble” should be read as “not gelled” (to apply similarlyhereinafter).

In addition, although said compound (d) may be soluble or insoluble insolvent (N), a cured product of said compound (d) preferably gels whenit is a crosslinked polymer in order to efficiently arrange functionalgroups possessed by said compound (d) on the pore surface, and ispreferably insoluble in case the cured product of compound (d) is anon-crosslinked polymer to avoid running off.

Linear polymer (Q) is also arbitrary, and polymer listed as an exampleof the aforementioned linear polymer (P), for example, can be used. Theuse of a mixture of two or more types of linear polymer (Q) ispreferable since the pore diameter of the surface of the porous resinlayer having a three-dimensional mesh structure does not becomeexcessively small, and it is difficult for macrovoids to form inside theporous resin layer having a three-dimensional mesh structure.

Although solvent (W) may or may not dissolve or swell a polymer formedfrom activating energy beam-polymerizeable compound (d), dissolving orswelling said polymer is preferable since functional groups can be fixedat high density on the pore surface. A solvent can be used for solvent(W) by suitably selecting from the solvents listed as examples of theaforementioned solvent (S).

Although solvent (N) preferable does not dissolve a polymer formed fromactivating energy beam-polymerizeable compound (d) in the case it is anon-crosslinked polymer, in the case said polymer is a crosslinkedpolymer, it may or may not swell said polymer. A solvent can be used forsolvent (N) by suitably selecting from the solvents listed as examplesof the aforementioned solvent (T).

In the present fourth method, in the case of forming a porous resinlayer having a three-dimensional mesh structure in a limited region orplurality of regions, said porous resin layer can be formed on a portionof a substrate by, for example, a method in which membrane depositionliquid (L) is only coated at the portion where said porous resin layeris formed by screen printing and so forth, or a method in which thoseportions other than where said porous resin layer is formed are coveredwith masking tape.

The present fourth method allows functional groups to be easilyintroduced at high density on the pore surface of a porous resin layerhaving a three-dimensional mesh structure by using an activating energybeam-polymerizeable compound (d) having a functional group.

A porous resin layer having a three-dimensional mesh structure capableof being formed by the aforementioned examples of methods may be formedover the entire side of a substrate or on a portion of a substrate. Inthe case of the latter, a channel may be formed that passes through botha portion where said porous resin layer is formed and a portion where itis not formed. As a result, a microfluidic device can be formed that hasa porous resin layer portion having a three-dimensional mesh structureat a portion of the bottom of the channel, but does not have said porousresin layer at other portions. At this time, by making the planardimensions of the porous resin layer having a three-dimensional meshstructure formed on a portion of a substrate larger than the planardimensions of the channel, it is not necessary to precisely align thelocations of said porous resin layer and channel, thereby facilitatingproduction. The porous resin layer having a three-dimensional meshstructure at a location other than the channel portion is filled with acured product of activating energy beam-curable resin composition (X).For example, by forming said porous resin layer in n number of parallellines (where n is a positive integer) on a substrate, and forming thechannel oriented perpendicular to them, spots of a porous resin layerhaving a three-dimensional mesh structure can be formed at n locationswithin the channel without requiring precise alignment.

There are no particular limitations on the shape of the substrate usedin the aforementioned examples of methods, and a substrate can be usedthat has an arbitrary shape according to the purpose of use. Althoughexamples of such shapes include sheets (including films, ribbons andbelts), plates, rolls and spheres, the coated surface is preferablyplanar or has a two-dimensional curvature from the viewpoint offacilitating coating of composition (X) thereon and facilitatingirradiation with an activating energy beam.

The substrate may also be surface-treated in the case of a polymer orother material. Examples of the purposes of surface treatment includepreventing dissolution by the membrane deposition liquid in the reactioninduction-type phase separation method or wet method, improvement ofwettability of the membrane deposition liquid, and improvement ofadhesion of the porous resin layer having a three-dimensional meshstructure.

The method for surface treatment of the substrate is arbitrary, examplesof which include treatment in which a composition containing a compoundselected from the group of compounds listed as examples ofpolymerizeable compound (a) is coated onto the surface of the substrateand then cured by irradiating with an activating energy beam, coronatreatment, plasma treatment, flame treatment, acid or base treatment,sulfonation treatment, fluorination treatment, primer treatment with asilane coupling agent and so forth, surface graft polymerization,coating with a surfactant or mold releasing agent, and physicaltreatment such as rubbing or sandblasting.

According to the aforementioned examples of methods, a porous resinlayer having a sponge-like structure, aggregated particle-likestructure, structure having macrovoids or structure consisting of amixture of these shapes, can be formed on the surface of a substrate. Inaddition, since the resulting porous resin layer having athree-dimensional mesh structure has a large surface area in the form ofthe surfaces of a large number of pores, it is able to fix a largeamount of catalyst, enzyme, DNA, sugar chains, cells or proteins.

If the surface of a porous resin layer having a three-dimensional meshstructure is made to be hydrophobic, enzymes, antigens and otherproteins can be fixed to the surface of the porous resin layer having athree-dimensional mesh structure by hydrophobic interactions withouthaving to introduce functional groups onto the porous surface. On theother hand, in the case of fixing proteins, DNA or sugar chains and soforth, by introducing a reactive functional group (such as an aminogroup, carboxyl group, hydroxyl group, epoxy group, aldehyde group,isocyanate group or —COCl group) onto the pore surfaces of the porousresin layer having a three-dimensional mesh structure in advance, andthen reacting an amino group, hydroxyl group, phosphate group orcarboxyl group of the aforementioned protein, DNA or sugar chain eitherdirectly or mediated by another functional group, the protein, DNA orsugar chain and so forth can be fixed to the surface of the porous resinlayer having a three-dimensional mesh structure by covalent bonding.

The thickness of the porous resin layer having a three-dimensional meshstructure should be suitably selected according to the purpose of use.For example, in the case of using in affinity chromatography, thethickness of said porous resin layer is preferably 3 to 100 μm, and morepreferably 5 to 50 μm.

The surface of the resulting porous resin layer having athree-dimensional mesh structure may be surface-treated using a methodpreviously listed as an example of substrate surface treatmentcorresponding to the application. For example, the surface of saidporous resin layer can be treated by a method such as coating acomposition containing one or more types of compounds selected from thegroup of compounds listed as examples of polymerizeable compound (a)(and particularly hydrophilic and amphiphilic polymerizable compounds)onto the surface of said porous resin layer and curing by irradiatingwith an activating energy beam for the purpose of introducing ahydrophilic group, hydrophobic group and other functional groups ontothe surface of said porous resin layer for the purpose of inhibitingnon-specific absorption of a solute such as protein or DNA onto saidporous resin layer surface.

In step (2), an uncured coating of composition (X) is formed inside aporous resin layer having a three-dimensional mesh structure and on saidporous resin layer by coating composition (X) onto said porous resinlayer to impregnate composition (X) inside said porous resin layer.Subsequently, an activating energy beam is radiated onto the uncuredcoating at those locations other than where a channel is to be formed toremove uncured composition (X) at the non-irradiated portions. As aresult, an indentation is obtained in which the bottom surface iscomposed of said porous resin layer while the wall surface is composedof a cured or semi-cured coating of composition (X), and the pores ofsaid porous resin layer at locations other than the portion serving asthe channel are obstructed by a cured or semi-cured product of theimpregnated composition (X).

Activating energy beam-polymerizeable compound (a) used in step (2) (tobe referred to as polymerizeable compound (a)) is a compound that isable to be polymerized by an activating energy beam in the presence orabsence of a polymerization initiator, and preferably is an additionpolymerizeable compound or has polymerizeable carbon-carbon double bondsfor the activating energy beam-polymerizeable functional groups. Ahighly reactive (meth)acrylic compound, vinyl ethers and maleimide-basedcompounds that are cured even in the absence of a photopolymerizationinitiator are particularly preferable.

In addition, if polymerizeable compound (a) is a multifunctionalcompound, strength after curing is also enhanced since a crosslinkedstructure is formed due to polymerization.

A compound similar to polymerizeable compound (b) able to be used in theaforementioned reaction induction-type phase separation method, forexample, can be used for this polymerizeable compound (a).

Polymerizeable compound (a) can be used alone or as a mixture of two ormore types. In addition, it may also be used after mixing with amonofunctional monomer in order to adjust viscosity or impart functionssuch as adhesiveness, stickiness or hydrophilicity.

A compound similar to the monofunctional monomers able to be used in theaforementioned reaction induction-type phase separation method, forexample, can be used as a mixable monofunctional monomer.

Composition (X) contains at least the aforementioned polymerizeablecompound (a). In addition to polymerizeable compound (a), saidcomposition (X) preferably also contains amphiphilic polymerizablecompound capable of copolymerizing with polymerizeable compound (a)(said amphiphilic polymerizable compound is to be referred to asamphiphilic compound (c)). As a result of composition (X) containingamphiphilic compound (c), the resulting cured product can be made to beresistant to swelling in water, and the surface of the cured product canbe made to be hydrophilic and have low adsorbency with respect tobiological components.

A compound that contains both hydrophilic groups and hydrophobic groupswithin its molecule, and has a polymerizeable compound capable ofcopolymerizing with activating energy beam-polymerizeable compound (a)contained in composition (X) by irradiating with an activating energybeam, can be used for amphiphilic compound (c). Although it is notnecessary for amphiphilic compound (c) to become a crosslinked polymer,a compound that becomes a crosslinked polymer may be used. In addition,amphiphilic compound (c) should be that which is uniformly compatiblewith polymerizeable compound (a). Here, compatibility refers to notcausing macroscopic phase separation, and includes a state in whichmicelles are formed and stably dispersed.

In the case polymerizeable compound (a) is a compound having two or morepolymerizeable carbon-carbon unsaturated bonds in its molecule,amphiphilic compound (c) is preferably a compound having one or morepolymerizeable carbon-carbon unsaturated bonds in its molecule.

Amphiphilic compound (c) is a compound that has a hydrophilic group andhydrophobic group in its molecule, and is compatible with water orhydrophobic solvent, respectively. In this case as well, compatibilityrefers to not causing macroscopic phase separation, and includes a statein which micelles are formed and stably dispersed.

Amphiphilic compound (c) preferably has solubility in water of 0.5% byweight or more at 0° C., and solubility in a mixed solvent ofcyclohexane and toluene (5:1, weight ratio) at 25° of 25% by weight ormore. Solubility referred to here indicates, for example, at least 0.5%by weight of the compound is able to be dissolved in the case ofsolubility of 0.5% by weight or more. If a compound is used for which atleast solubility in water or solubility in cyclohexane and toluene (5:1,weight ratio) is lower than these values, it is difficult to obtain acured product having superior characteristics for both surfacehydrophilicity and hydrophobicity.

In the case amphiphilic compound (c) has a nonionic hydrophilic group,and particularly a polyether-based hydrophilic group, the balancebetween hydrophilicity and hydrophobicity in terms of Griffin's HLBvalue is preferably within the range of 11 to 16, and more preferablywithin the range of 11 to 15. If outside this range, it either becomesdifficult to obtain a molded product having high hydrophilicity andsuperior moisture resistance, or the combination or mixing ratio ofcompounds for obtaining this ends up being limited.

The hydrophilic group possessed by amphiphilic compound (c) isarbitrary, and examples of which include cationic groups such as anamino group, quaternary ammonium group or phosphonium group; anionicgroups such as a sulfone group, phosphoric acid group or carbonyl group;nonionic groups such as a hydroxyl group, polyethylene glycol chain oramide group; and amphoteric groups such as an amino acid residue.Amphiphilic compound (c) is preferably a compound having a polyethergroup as a hydrophilic group, and is particularly preferably a compoundhaving 6 to 20 repeating polyethylene glycol chains.

Examples of hydrophobic groups of amphiphilic compound (c) include alkylgroups, alkylene groups, alkylphenyl groups, long-chain alkoxy groups,fluorine-substituted alkyl groups and siloxane groups. Amphiphiliccompound (c) is preferably a compound having an alkyl group or alkylenegroup having 6 to 20 carbon atoms as a hydrophobic group. Alkyl oralkylene groups having 6 to 20 carbon atoms may be contained in the formof, for example, alkylphenyl groups, alkylphenoxy groups, alkoxy groupsor phenylalkyl groups.

Amphiphilic compound (c) is preferably a compound having 6 to 20repeating polyethylene glycol chains as a hydrophilic group, and analkyl or alkylene group having 6 to 20 carbon atoms as a hydrophobicgroup. Among these amphiphilic compounds (c), nonylphenoxy polyethyleneglycol (n=8-17) (meth)acrylate and nonylphenoxy polypropylene glycol(n=8-17) (meth)acrylate are particularly preferable.

Although differing according to the types and combination ofpolymerizeable compound (a) and amphiphilic compound (c), the preferableratio of polymerizeable compound (a) and amphiphilic compound (c)contained in composition (X) is preferably 0.1 to 5 parts by weight, andmore preferably 0.2 to 3 parts by weight, of amphiphilic compound (c) to1 part by weight of polymerizeable compound (a). If the amount ofamphiphilic compound (c) is less than 0.1 part by weight with respect to1 part by weight of polymerizeable compound (a), it becomes difficult toform a highly hydrophilic surface, while if the amount of amphiphiliccompound (c) exceeds 5 parts by weight, composition (X) swells in waterand a polymer of composition (X) has the risk of gelling.

A cured product that demonstrates high hydrophilicity and lowadsorptivity without gelling in a swollen state can be produced bysuitably selecting the mixing ratio of polymerizeable compound (a) andamphiphilic compound (c). It is preferable to reduce the amount ofamphiphilic compound (c) added the stronger the degree of hydrophilicityof amphiphilic compound (c), or for example, the larger Griffin's HLBvalue.

A polymerizeable compound (b) having a functional group to be introducedonto the surface of a porous resin layer having a three-dimensional meshstructure is preferably mixed into composition (X). In the case ofintroducing a certain functional group onto the surface of a porousresin layer having a three-dimensional mesh structure in particular, aresin having said functional group is preferably used for the resin thatforms the porous resin layer having a three-dimensional mesh structure,and a polymerizeable compound (b) having said functional group ispreferably mixed into composition (X). As a result, in a step to bedescribed later in which uncured composition (X) at a non-irradiatedportion is removed, even if the removal of composition (X) isincomplete, the problem of the pore surfaces being covered byprecipitates of said composition thereby causing said functional groupto not be exposed can be prevented.

In addition, a photopolymerization initiator, polymerization retardant,polymerization inhibitor, solvent, thickener, modifier or colorant andso forth can be mixed into composition (X).

Compounds similar to the photopolymerization initiators, polymerizationretardants and polymerization inhibitors of membrane deposition liquid(J) in the aforementioned reaction induction-type phase separationmethod can be preferably used as photopolymerization initiators,polymerization retardants and polymerization inhibitors able to be addedto composition (X).

Although there are no particular limitations on the solvent, it isnecessary to suitably adjust the type and amount of solvent addedaccording to additives added to the polymerizeable compound (a) andcomposition (X) used or according to the required viscosity and soforth. Examples of such solvents include alcohols such as ethanol,ketones such as acetone, amide-based solvents such asN,N-dimethylformamide, and chlorine-based solvents such as methylenechloride.

Although the viscosity of composition (X) can be changed according tothe pore diameter of the porous resin layer having a three-dimensionalmesh structure, the viscosity of composition (X) at 25° C. is preferablywithin the range of 30 to 3000 mPa·s, and more preferably within therange of 100 to 1000 mPa·s, from the viewpoint of allowing composition(X) to rapidly penetrate into the porous resin layer having athree-dimensional mesh structure when coating over the porous resinlayer having a three-dimensional mesh structure, and composition (X)being completely removed from the porous resin layer having athree-dimensional mesh structure during removal of uncured composition(X) at a non-irradiated portion. If the viscosity of composition (X) isless than 30 mPa·s, it becomes difficult to control the depth of theindentation, and if the viscosity exceeds 3000 mPa·s, it becomesdifficult for composition (X) to penetrate into the porous resin layerhaving a three-dimensional mesh structure, and removal of uncuredcomposition (X) at a non-irradiated portion also becomes difficult.

In step (2), an arbitrary coating method can be used to coat composition(X) onto a porous resin layer having a three-dimensional mesh structure,examples of which include spin coating, roller coating, spreading,dipping, spraying, using a bar coater, using an X-Y applicator, screenprinting, relief printing, gravure printing, extrusion from a nozzle andmold casting. In addition, in the case composition (X) has highviscosity or is particularly thinly coated, composition (X) can becoated by a method in which a solvent is contained in composition (X)and said solvent is volatilized after coating.

Although there are no particular limitations on the thickness at whichcomposition (X) is coated provided that a cured or semi-cured coating isobtained on the upper portion of the surface of a porous resin layerhaving a three-dimensional mesh structure after irradiating with anactivating energy beam, in the case of fixing a specific substance onsaid porous resin layer on a bottom surface and using for affinitychromatography, the thickness of a cured or semi-cured coating formed onthe upper portion of said porous resin layer after irradiating with anactivating energy beam, namely the wall height of an indentation, ispreferably within the range of 3 to 150 μm, and more preferably withinthe range of 5 to 50 μm. If the thickness is less than 3 μm, there isthe risk of the channel being obstructed when another member serving asa cover is adhered to the indentation to allow said indentation to serveas a cavity-like channel. On the other hand, if the thickness is greaterthan 150 μm, it becomes difficult for substances in an aqueous solutionto be adsorbed to and released from (interact with) a porous resin layerhaving a three-dimensional mesh structure on the bottom surface of achannel while the aqueous solution passes through the channel, therebymaking this unsuitable for affinity chromatography applications.

In the case of adding a solvent to activating energy beam-curable resincomposition (X), said solvent is removed by volatilization followingcoating. The method of removal is arbitrary, and examples of methodsthat can be used include air drying, hot air drying, infrared lightdrying, vacuum drying and microwave drying. Although solvent may beremoved after irradiating with an activating energy beam, it ispreferably removed prior to irradiation with the activating energy beamin order to accurately control the dimensions and shape of the channel.

Examples of radiated activating energy beams include light rays such asultraviolet rays, visible light rays, infrared rays, laser light raysand radiant light rays; ionizing radiation such as X-rays, gamma raysand radiant light rays; and particle beams such as electron beams, ionbeams, beta rays and heavy particle beams. Among these, ultraviolet raysor visible light is preferable in terms of handling ease and curingrate, while ultraviolet rays are particularly preferable. Irradiationwith an activating energy beam is preferably carried out in a low oxygenconcentration atmosphere for the purpose of accelerating curing rate andensuring complete curing. Preferable examples of low oxygenconcentration atmospheres include a vacuum or reduced pressureatmosphere in the presence of flowing nitrogen, flowing carbon dioxideor flowing argon.

The aforementioned activating energy beam is radiated in a pattern whenradiating the activating energy beam in order to form an indentation inwhich a porous resin layer having a three-dimensional mesh structure isformed on all or a portion of the bottom surface. The method employedfor patterning radiation is arbitrary, and examples of methods that canbe used include radiating the activating energy beam after masking theportion that is not to be irradiated with the activating energy beam,and photolithography involving scanning with a laser or other activatingenergy beam. In the case of using a photomask, the photomask may be of anon-contact type or contact type with the coating.

Adhesion with another member serving as a cover is possible withoutusing an adhesive by curing or semi-curing an uncured coating ofcomposition (X), or adhesive strength is improved in the case of usingan adhesive. In the case composition (X) is in a semi-cured state,although it is preferable to completely cure composition (X) by carryingout post-curing in any step prior to obtaining the final microfluidicdevice, composition (X) is not required to be completely cured providedit does not impair the function of a microfluidic device of the presentinvention. In the case of curing using an activating energy beam,post-curing may use the same or different activating energy beam as thatused for semi-curing. Post-curing may also be carried out by heat curingin addition to curing by an activating energy beam.

In step (3), another member serving as a cover is adhered to theindentation of a member having an indentation formed in step (2) toallow the aforementioned indentation to serve as a cavity-like channel.

The member that serves as a cover can be suitably selected according tothe purpose of use, a member should be used that is not attacked by theliquid that flows through the channel, and said member may be in theform of a tape, sheet or plate having stickiness.

A cover member and a member having an indentation should be laminated inorder to cover the indentation with the member serving as a cover. Aspreviously described, the member having an indentation may be laminatedas is if it is a semi-cured coating and has satisfactory adhesivenesswith the cover member. In addition, in the case the adhesiveness of themember having an indentation is low or is a cured coated film, bothmembers should be laminated using an adhesive and so forth.

In addition, a method may also be employed in which a compositioncontaining an activating energy beam-polymerizeable compound is coatedonto a substrate such as a polymer film or sheet, the substrate isirradiated with an activating energy beam to semi-cure the coating ofsaid composition, and the substrate is laminated onto an indication of amember having the aforementioned indentation followed by againirradiating with an activating energy beam to completely cure the coatedfilm of said composition. The activating energy beam-polymerizeablecompound and its composition used here may be the same as polymerizeablecompound (a) and composition (X) used in the aforementioned step (2),and may one or more types of compounds selected from the group ofcompounds listed as examples of compounds that can be used forpolymerizeable compound (a). Coating of the polymerizeable compoundshould also be in compliance with step (2).

Examples of adhesives that can be used as adhesives when laminating thecover member and member having an indentation include epoxy resin-basedadhesives, styrene butadiene resin-based adhesives and (meth)acrylicadhesives.

The use of a production process of the present invention makes itpossible to easily obtain a microfluidic device having a porous resinlayer having a three-dimensional mesh structure of uniform thickness onthe inner surface of a minute channel without obstructing said channel.In addition, according to said production process, since a plurality ofmicrofluidic devices can be easily fabricated on a single substrate(exposed developing plate) without requiring positioning, a large numberof microfluidic devices can be produced all at once with satisfactoryreproducibility and superior dimensional stability.

Among microfluidic devices obtained according to the aforementionedprocess, a microfluidic device that forms a cavity by (I) having aporous resin layer having a three-dimensional mesh structure on theupper portion of a substrate, (II) filling said porous resin layer witha cured resin of an activating energy beam-curable composition (X)impregnated except for a channel portion, and (III) having a porousresin layer having a three-dimensional mesh structure in which thechannel is not filled with a cured resin of activating energybeam-curable composition (X), a cured resin layer of activating energybeam-curable composition (X) formed in the upper portion of a porousresin layer having a three-dimensional mesh structure filed with a curedresin of activating energy beam-curable composition (X), and a cover,serving as wall surfaces, can be used particularly preferable for amicrofluidic device composed of a substrate, porous resin layer, channeland cover.

The size of the aforementioned channel cross-section should be a sizethat allows interaction between a separation target substance containedin the fluid flowing through the channel and the porous resin layerhaving a three-dimensional mesh structure, and in the case of a channelcross-section at a portion of the channel having the porous resin layerhaving a three-dimensional mesh structure, if an arbitrary point in saidcross-section is designated as x, the portion of the porous resin layerhaving a three-dimensional mesh structure that is closest to saidarbitrary point in terms of straight line distance is designated as y,the straight line distance between x and y is designated as r, and themaximum distance that r is able to have in said cross-section isdesignated as r_(max), then the microfluidic device should be designedso that said r_(max) is within the range of 1 to 50 μm. If the value ofr_(max) defined here is 50 μm or less, a separation target substancecontained in the liquid that flows through the channel is able tosatisfactorily interact with the porous resin layer having athree-dimensional mesh structure on the inner walls of the channel. Inaddition, if the value of r_(max) is 1 μm or more, then the pressurerequired to cause the flow of the liquid does not become excessivelylarge.

The microfluidic device should be suitably designed according to thearrangement of the porous resin layer having a three-dimensional meshstructure and the cross-sectional shape of the channel so that the valueof r_(max) in the channel cross-section is within the aforementionedrange. For example, in the case of a channel having a porous resin layerhaving a three-dimensional mesh structure on the bottom and top of theinner walls and having a rectangular or trapezoidal cross-section, theheight of the channel cross-section should be 100 μm or less, while inthe case of a channel having a porous resin layer having athree-dimensional mesh structure only one surface of the inner walls andhaving a rectangular or trapezoidal cross-section, the distance betweensaid porous resin layer and the side opposing said porous resin layershould be 1 to 50 μm. In the case of a channel having a circular ortriangular cross-section, the microfluidic device should be similarlysuitable designed according to the porous resin layer having athree-dimensional mesh structure of the inner walls. Furthermore, in thepresent invention, a rectangular or trapezoidal cross-section includes ashape having rounded corners.

On the other hand, the ratio between the maximum width and maximumheight of the channel cross-section, as represented in the form of theratio of maximum width to maximum height (maximum width/maximum height),is preferably within the range of 1/20 to 20/1, and particularlypreferably within the range of 1/10 to 10/1, in terms of the ease ofoptically reading the spot of an analysis target substance flowingthrough the channel, the ease of controlling the flow rate, temperatureand so forth, and the ease of production.

Channel length is arbitrary, and although a suitable length can beadopted according to the purpose of use, it is preferably 1 to 500 mmand more preferably 5 to 200 mm. If the channel length is greater thanor equal to the aforementioned lower limit, adequate separationperformance can be obtained, and if the channel length is less than orequal to the aforementioned upper limit, the required liquid pumpingrate can be decreased, separation time can be shortened, and the size ofthe channel can be reduced.

In addition, the form of the direction parallel to the direction ofliquid flow through the channel is arbitrary, and may be linear, curvedor a combination thereof or branched. The width of the channel is alsonot required to be constant. In addition, the locations and quantity ofchannel openings are also arbitrary, and a single channel may have aplurality of openings. In addition, the number of independent channelspresent in a single member is also arbitrary.

A channel of a microfluidic device of the present invention has a porousresin layer having a three-dimensional mesh structure on one of itsinner wall surfaces or on two opposing surfaces.

A non-porous member can be used as a cover for forming a porous resinlayer having a three-dimensional mesh structure on one surface inside achannel. A member in which a porous resin layer having athree-dimensional mesh structure is formed on a substrate as previouslydescribed, or a member having a groove on said porous resin layer, forexample, can be used as a cover to form a porous resin layer having athree-dimensional mesh structure on two opposing surfaces of channelinner walls.

The aforementioned porous resin layer having a three-dimensional meshstructure is formed on one surface or two opposing surfaces of the wallsof a channel, and an analysis target substance that flows through thechannel is separated as a result of moving through the channel whileinteracting with said porous resin layer. In addition, in the case of aprobe being fixed to said porous resin layer, a target analysissubstance is separated as a result of moving through the channel whileinteracting with said probe.

The formed site of a porous resin layer having a three-dimensional meshstructure in a direction parallel to the direction of movement of liquidthrough a channel may be the entire channel or partway through thechannel. In the case the present microfluidic device is, for example, achromatography device or electrophoresis analysis device, forming saidporous resin layer continuously and without interruption in the channelis preferable for improving separation performance.

The thickness of the aforementioned porous resin layer having athree-dimensional mesh structure is preferably 0.5 to 30 μm, morepreferably 1 to 20 μm, and most preferably 2 to 10 μm. By making thethickness equal to or greater than this lower limit, the surface areafor interaction with an analysis target substance is sufficiently large,and separation performance is enhanced. On the other hand, making thethickness less or equal to the upper limit prevents a separation targetsubstance in solution from penetrating inside deep pores causing adecrease in the migration rate, thereby making it possible to shortenseparation and analysis times.

Although the pore diameter of the pores of the aforementioned porousresin layer having a three-dimensional mesh structure is arbitrary, itis preferably 0.05 to 3 μm, and more preferably 0.1 to 1 μm. By makingthe pore diameter equal to or greater than this lower limit, an adequateamount of probe can be fixed. In addition, by making the pore diameterless than or equal to the upper limit, since macromolecules likeproteins can also be fixed as probes, the substance migration ratebetween the deep portion and surface of the porous resin layer having athree-dimensional mesh structure increases, thereby allowing rapidseparation. Furthermore, the aforementioned pore diameter is the porediameter of a large number of pores, and does not necessarily refer tothe mean diameter. A narrow distribution of the aforementioned porediameter is preferable since it results in higher separation efficiency.

Although the porosity of the aforementioned porous resin layer having athree-dimensional mesh structure is arbitrary, it is preferably 30 to90%, and more preferably 40 to 70%. If porosity is made to be withinthis range, surface area can be adequately increased without causing adecrease in mechanical strength.

The previously described organic polymers can be used for the materialof the aforementioned porous resin layer having a three-dimensional meshstructure, and the material is more preferably an activating energybeam-curable resin since it can be molded easily. In the case of fixinga probe, although a material that facilitates probe fixation can bearbitrarily selected, it is preferably an organic polymer sinceproduction is easy, and it is more preferably an activating energybeam-curable resin since it can be molded easily.

In addition, arbitrary functional groups can be introduced into theaforementioned porous resin layer having a three-dimensional meshstructure. Examples of hydrophilic functional groups include nonionicfunctional groups such as hydroxyl groups, polyethylene glycol groups,amide groups and nitro groups, anionic functional groups such ascarboxyl groups, sulfone groups, phosphoric acid groups, phosphorousacid groups, (substituted) hydroxyphenyl groups and silanol groups, andcationic functional groups such as (N-substituted) amino groups,quaternary ammonium groups, phosphonium groups and sulfonium groups.Examples of hydrophobic functional groups include fluorine, chlorinegroups, siloxane structures, alkyl groups and phenyl groups. Examples ofamphoteric functional groups include amino acid residues. Other examplesof functional groups include amphiphilic functional groups. Otherexamples also include photoreactive groups such as azides. Furthermore,these functional groups may be made to function themselves such as byimparting selective adsorptivity, they can be converted into activefunctional groups by a chemical reaction, or they can be used as anchorsfor fixing (bio)chemical substances or biological substances and soforth.

Other substances are also preferably fixed to the aforementioned porousresin layer having a three-dimensional mesh structure. Examples of othersubstances include various types of catalysts, enzymes, antibodies,antigens and other proteins, oligonucleotides such as DNA and RNA,sugar-containing substances such as sugar chains and glycolipids,biological tissue such as cell membranes, organelle and cells, andliving organisms such as bacteria. These substances may naturally alsobe chemically modified. In the case of using an oligonucleotide as afixed probe in particular, genes can be separated and detected, therebyallowing these substances to be used effectively for the detection ofsingle-base gene mutations.

In the case of using an oligonucleotide as a probe, the length of theoligonucleotide is preferably 5 to 30 nucleotides, more preferably 5 to20 nucleotides, and most preferably 5 to 10 nucleotides. By making thelength of the oligonucleotide within this range, a sufficiently reliableand high separation rate can be obtained at a temperature that allowsthe present invention to be carried out easily, namely from roomtemperature to 60° C. In addition, it is also preferable for the samepurpose to use an oligonucleotide comprised of a nucleotide sequencethat intentionally contains mismatches with respect to a polynucleotideor oligonucleotide targeted for analysis for the probe oligonucleotide.

In the case of using a microfluidic device of the present invention as aliquid chromatography column, the amount of probe (including functionalgroups) fixed to the surface of the channel is preferably in excess withrespect to the amount of analysis target substance having affinity withthe probe. In the case the amount of probe is in excess, the amount ofanalysis target substance involved in interaction with the probeincreases as compared with only using an insufficient amount of probe,and since the local concentration of target analysis substance becomeshigher, the target analysis substance can be detected with highsensitivity. For reasons such as this, the greater the amount of probefixed to the porous resin layer having a three-dimensional meshstructure, the better.

In addition, in the case of using a microfluidic device of the presentinvention as a member for affinity electrophoresis as well, the amountof probe fixed is preferably large as in the aforementioned case ofliquid chromatography.

EXAMPLES

Although the following provides a more detailed explanation of thepresent invention using its examples, the present invention is notlimited to the scope of these examples. Furthermore, in the followingexamples, the terms “parts” and “%” respectively refer to “parts byweight” and “% by weight” unless indicated otherwise.

Measurement of viscosity and irradiation with an ultraviolet lamp in theexamples were carried out using the methods indicated below.

[Viscosity Measurement]

The viscosity of a composition at 25° was measured using the Model VDH-Kviscometer manufactured by Shibaura Systems Co., Ltd.

[Irradiation with Ultraviolet Lamp 1]

Ultraviolet light having an ultraviolet intensity of 40 mW/cm² at 365 nmwas radiated at room temperature in a nitrogen atmosphere unlessindicated otherwise using the Model UE031-353CHC UV Radiation Devicemanufactured by Eyegraphics Co., Ltd., which uses a 3000 W metal halidelamp for the light source.

[Irradiation with Ultraviolet Lamp 2]

Ultraviolet light having an ultraviolet intensity of 100 mW/cm² at 365nm was radiated at room temperature in a nitrogen atmosphere unlessindicated otherwise using the light source unit of the Multilight Model200 UV Exposure Device manufactured by Ushio Inc. which uses a 200 Wmetal halide lamp for the light source.

Example 1

The present example 1 is an example of producing a porous resin layerhaving a three-dimensional mesh structure according to the “reactioninduction-type phase separation method”.

[Preparation of Membrane Deposition Liquid (J)]

72 parts of a trifunctional urethane acrylate oligomer (Unidic V-4263,Dainippon Ink and Chemicals) having an average molecular weight of 2000,18 parts of dicyclopentanyl diacrylate (R-684, Nippon Kayaku), 10 partsof glycidyl methacrylate (Wako Pure Chemical Industries), 150 parts ofmethyl decanoate (Wako Pure Chemical Industries), 10 parts of a volatilegood solvent in the form of acetone, and 3 parts of an ultravioletpolymerization initiator in the form of 1-hydroxycyclohexyl phenylketone (Irgacure 184, Ciba-Geigy) were uniformly mixed to preparemembrane deposition liquid (J1).

[Preparation of Composition (X)]

50 parts of a trifunctional urethane acrylate oligomer (Unidic V-4263,Dainippon Ink and Chemicals) having an average molecular weight of 2000,40 parts of hexanediol diacrylate (New Frontier HDDA, DaiichiPharmaceutical), 10 parts of glycidyl methacrylate (Wako Pure ChemicalIndustries), 5 parts of a photopolymerization initiator in the form of1-hydroxycyclohexyl phenyl ketone (Irgacure 184, Ciba-Geigy) and 0.5parts of polymerization retardant in the form of2,4-diphenyl-4-methyl-1-pentene (Kanto Chemical) were mixed to preparecomposition (X1). The viscosity of said composition (X1) was 192 mPa·s.

[Step 1: Formation of Porous Resin Layer Having a Three-Dimensional MeshStructure]

Membrane deposition liquid (J1) was coated at a rotating speed of 600rpm using a spin coater (Mikasa) onto an acrylic sheet having athickness of 1 mm used as a substrate, and said deposition liquid (J1)was irradiated for 40 seconds with ultraviolet light from ultravioletlamp 1 to cure membrane deposition liquid (J1) followed by washing withn-hexane to remove poor solvent (R) to form a porous resin layer (1)having a three-dimensional mesh structure.

[Step 2: Formation of Indentation (Channel) in Which Porous Resin LayerHaving a Three-Dimensional Mesh Structure is Exposed on Bottom Surface]

Composition (X1) was coated at a rotating speed of 800 rpm using a spincoater (Mikasa) onto the aforementioned porous resin layer (1) having athree-dimensional mesh structure to form an uncured coating of saidcomposition (X1), said uncured coating was irradiated for 120 secondswith ultraviolet light from ultraviolet lamp 2 through a photomask atlocations other than where the channel is to be formed to form asemi-cured coating of said composition (X1), and the non-irradiatedportion of uncured composition (X1) was removed with ethanol to form anindentation (channel 1) on the substrate in which porous resin layer (1)having a three-dimensional mesh structure is exposed on the bottomsurface.

[DNA Fixation]

(Introduction of Amino Groups)

A 5% by weight aqueous solution of polyallylamine (molecular weight:15000, Nitto Boseki) was contacted with the indentation (channel 1)prepared in the aforementioned step 2 and allowed to react for 2 hoursat 50° C. (a portion of the amino groups in the polyallylamine werereacted with epoxy groups in the porous resin layer having athree-dimensional mesh structure), followed by washing for 15 minutesunder running water to introduce amino groups into said porous resinlayer.

(Introduction of Aldehyde Groups)

A substrate having the aforementioned indentation (channel 1) into whichamino groups were introduced was placed in a 5% by weight aqueoussolution of glutaraldehyde (Wako Pure Chemical Industries) and allowedto react for 2 hours at 50° C. (nearly all of the amino groups in thepolyallylamine were reacted with one of the aldehyde groups ofglutaraldehyde), followed by washing for 10 minutes under running waterto introduce aldehyde groups into the porous resin layer having athree-dimensional mesh structure.

(DNA Fixation)

One μL of an aqueous solution (concentration: 50 μM) of DNA having anamino-modified 5′ terminal and a fluorescein isothiocyanate isomer I(FICT-I)-modified 3′ terminal (length: 25 nucleotides, Espec OligoService) was dropped into the aforementioned indentation (channel 1) inwhich aldehyde groups were introduced, and after reacting for 15 minutesat 50° C. and humidity of 100% (the terminal amino groups of DNA wereallowed to react with the aldehyde groups of the porous resin layerhaving a three-dimensional mesh structure), the indentation was placedin a 0.2% by weight aqueous solution of sodium tetrahydroborate andallowed to undergo a reduction reaction for 5 minutes. Next, theindentation was rinsed with 0.2×SSC/0.1% SDS solution and then rinsedwith 0.2×SSC followed by additionally rinsing with distilled water andallowing to air dry to fix the DNA to the porous resin layer having athree-dimensional mesh structure on the bottom surface of theindentation (channel 1). (Here, 0.2×SSC refers to an aqueous solution of0.03 M NaCl and 3 mM sodium citrate, while 0.1% SDS refers to an aqueoussolution of 0.1% by weight sodium dodecyl sulfate.)

[Step 3: Adhesion of Cover]

A composition in which 72 parts of a trifunctional urethane acrylateoligomer (Unidic V-4263, Dainippon Ink and Chemicals) having an averagemolecular weight of 2000, 18 parts of hexanediol diacrylate (NewFrontier HDDA, Daiichi Pharmaceutical), 10 parts of glycidylmethacrylate (Wako Pure Chemical Industries) and 2 parts of aphotopolymerization initiator in the form of 1-hydroxycyclohexyl phenylketone (Irgacure 184, Ciba-Geigy) were uniformly mixed, was coated at arotating speed of 800 rpm using a spin coater (Mikasa) onto a biaxiallyoriented polypropylene film having a thickness of 30 μm subjected tocorona discharge treatment on one side (Futamura Chemical). Said uncuredcoating was irradiated for 1 second with ultraviolet light fromultraviolet lamp 1 to form a semi-cured film of the aforementionedcomposition. This was laminated over the indentation (channel 1)fabricated in the aforementioned step 2 and completely cured by againirradiating with ultraviolet light from ultraviolet lamp 1 for 40seconds to produce a microfluidic device (1) having a capillary-likechannel (channel 1) in which a porous resin layer having athree-dimensional mesh structure is exposed on the bottom surface.

[Observation of Structure of Porous Resin Layer Having aThree-Dimensional Mesh Structure]

When the surface of the aforementioned porous resin layer having athree-dimensional mesh structure fabricated in step 1 was observed witha scanning electron microscope, pores having a pore diameter of about0.4 μm were observed in the form of cavities between aggregatedparticles having a diameter of about 0.5 μm. In addition, when across-section of the porous resin layer having a three-dimensional meshstructure was observed, the thickness of said porous resin layer havinga three-dimensional mesh structure was about 10 μm. A scanning electronmicrograph of said porous resin layer having a three-dimensional meshstructure is shown in FIG. 1.

[Observation of Structure of Indentation (Channel)]

When a cross-section of the indentation (channel 1) fabricated in theaforementioned step 2 was observed with a scanning electron microscope,the cross-sectional shape of the indentation was rectangular having awidth of about 250 μm and a depth, excluding the porous resin layerhaving a three-dimensional mesh structure, of about 30 μm.

[DNA Quantification]

As a result of measuring the fluorescent intensity emitted by the FITC-Iin the porous resin layer, having a three-dimensional mesh structure onthe bottom surface to which the aforementioned DNA was fixed, with aFluoro Imaging Scanner (FLA-3000G, Fuji Photo Film), the value offluorescent intensity was 1069 LAU (units of fluorescent intensitydisplayed on the aforementioned instrument)/mm².

Example 2

The present Example 2 is an example of producing a porous resin layerhaving a three-dimensional mesh structure according to the “surfaceswelling method”.

[Preparation of Composition (X)]

A composition (X2) was prepared in the same manner as the preparation ofcomposition (X1) in Example 1 with the exception of mixing 40 parts of1,6-hexanediol ethoxylate diacrylate (Photomer 4361, Cognis Japan)instead of 40 parts of hexanediol diacrylate (New Frontier HDDA, DaiichiPharmaceutical), and mixing 10 parts of nonylphenoxy polyethylene glycol(n=17) acrylate (N-177E, Daiichi Pharmaceutical) instead of 10 parts ofglycidyl methacrylate (Wako Pure Chemical Industries). The viscosity ofthis composition was 220 mPa·s.

[Step 1: Formation of Porous Resin Layer Having a Three-Dimensional MeshStructure]

(Formation of Three-Dimensional Mesh Structure)

After immersing a polystyrene sheet (Dainippon Ink and Chemicals) havinga thickness of 150 μm used as a substrate in N,N-dimethylacetoamide(Wako Pure Chemical Industries) for 5 seconds at room temperature, thesheet was placed in water and additionally washed for about 5 minutesunder running water to obtain a polystyrene sheet in which a porousresin layer having a three-dimensional mesh structure and substrate wereintegrated into a single unit.

(Introduction of Epoxy Groups)

Moreover, a composition in which was mixed 2.5 parts of a trifunctionalurethane acrylate oligomer (Unidic V-4263, Dainippon Ink and Chemicals)having an average molecular weight of 2000, 2 parts of 1,6-hexanediolethoxylate diacrylate (Photomer 4361, Cognis Japan), 0.5 parts ofglycidyl methacrylate (Wako Pure Chemical Industries), 0.25 parts of aphotopolymerization initiator in the form of 1-hydroxycyclohexyl phenylketone (Irgacure 184, Ciba-Geigy) and 95 parts of a solvent in the formof ethanol, was coated at a rotating speed of 1500 rpm using a spincoater (Mikasa) onto said porous resin layer having a three-dimensionalmesh structure, followed by irradiating for 40 seconds with ultravioletlight from ultraviolet lamp 1 to form a porous resin layer (2) having athree-dimensional mesh structure in which epoxy groups are introducedonto the pore surfaces.

[Step 2: Formation of Indentation (Channel) in Which Porous Resin Layeris Exposed on Bottom Surface]

An indentation (channel 2) in which a porous resin layer having athree-dimensional mesh structure is exposed on the bottom surface wasformed using the same method as Example 1 with the exception of usingcomposition (X2) instead of composition (X1).

[DNA Fixation]

Introduction of amino groups and aldehyde groups, and fixation of DNAwere carried out in the same manner as Example 1.

[Step 3: Adhesion of Cover]

A cover was adhered to the indentation (channel 2) using the samecomposition and same method as in the case of Example 1 to produce amicrofluidic device (2) having a capillary-like channel (channel 2) inwhich a porous resin layer having a three-dimensional mesh structure isexposed on the bottom surface.

[Observation of Structure of Porous Resin Layer Having aThree-Dimensional Mesh Structure]

When the surface of the aforementioned porous resin layer (2) having athree-dimensional mesh structure fabricated in step 1 was observed witha scanning electron microscope, sponge-like pores having a pore diameterof about 0.8 μm were observed. In addition, when a cross-section of saidporous resin layer was observed, the thickness of said porous resinlayer was about 2 μm.

[Observation of Structure of Indentation (Channel)]

When a cross-section of the indentation (channel 2) fabricated in theaforementioned step 2 was observed with a scanning electron microscope,the cross-sectional shape of the indentation was rectangular having awidth of about 250 μm and a depth, excluding the porous resin layerhaving a three-dimensional mesh structure, of about 30 μm.

[DNA Quantification]

DNA was fixed to the indentation (channel 2) formed in theaforementioned step 2 using the same method as the DNA fixation methodindicated in the aforementioned Example 1. As a result of measuring thefluorescent intensity emitted by FITC-I fixed to said indentation(channel 2) using the same method as the DNA quantification of Example1, the value of fluorescent intensity was 954 LAU/mm².

Example 3

The present example 3 is an example of producing a porous resin layerhaving a three-dimensional mesh structure according to the “wet method”.

[Preparation of Membrane Deposition Liquid (K)]

5 parts of a linear polymer in the form of an aromatic polyamide (Conex,Teijin), 90 parts of a solvent (U) in the form of N,N-dimethylacetoamide(Wako Pure Chemical Industries), and 5 parts of an additive in the formof ethylene glycol were uniformly mixed to obtain membrane depositionliquid (K).

[Preparation of Composition (X)]

50 parts of tritetraethylene glycol bis-maleimide (Lumicure MIA200,Dainippon Ink and Chemicals), 40 parts of 1,6-hexanediol ethoxylatediacrylate (Photomer 4361, Cognis Japan), 10 parts ofN,N-dimethylacrylamide (DMAA, Kohjin), and 0.5 parts of polymerizationretardant in the form of 2,4-diphenyl-4-methyl-1-pentene (KantoChemical) were mixed to prepare composition (X3). The viscosity of saidcomposition (X3) was 100 mPa·s.

[Step 1: Formation of Porous Resin Layer Having a Three-Dimensional MeshStructure]

(Formation of Three-Dimensional Mesh Structure)

Membrane deposition liquid (K) was coated using a 50 μm bar coater ontoan acrylic sheet having a thickness of 1 mm used as a substrate, andsaid substrate was immersed in water to obtain a milky white, coagulatedcoating. The resulting coagulated coating was additionally washed for 10minutes under running water and then dried for 1 hour in a vacuum at 40°C. to obtain a porous resin layer having a three-dimensional meshstructure.

[Step 2: Formation of Indentation (Channel) in Which Porous Resin LayerHaving a Three-Dimensional Mesh Structure is Exposed on Bottom Surface]

An indentation (channel 3) in which porous resin layer (3) having athree-dimensional mesh structure is exposed on the bottom surface wasformed using the same method as Example 1 with the exception of usingcomposition (X3) instead of composition (X1).

(DNA Fixation)

The introduction of amino and aldehyde groups as well as DNA fixationwere carried out in the same manner as Example 1.

[Step 3: Adhesion of Cover]

A cover was adhered to the indentation (channel 3) using the samecomposition and same method as in the case of Example 1 to produce amicrofluidic device (3) having a capillary-like channel (channel 3) inwhich a porous resin layer having a three-dimensional mesh structure isexposed on the bottom surface.

[Observation of Structure of Porous Resin Layer Having aThree-Dimensional Mesh Structure]

When the surface of the aforementioned porous resin layer (3) having athree-dimensional mesh structure fabricated in step 1 was observed witha scanning electron microscope, sponge-like pores having a pore diameterof about 0.6 μm were observed. In addition, when a cross-section of saidporous resin layer was observed, the thickness of said porous resinlayer was about 35 μm.

[Observation of Structure of Indentation (Channel)]

When a cross-section of the indentation (channel 3) fabricated in theaforementioned step 2 was observed with a scanning electron microscope,the cross-sectional shape of the indentation was rectangular having awidth of about 250 μm and a depth, excluding the porous resin layerhaving a three-dimensional mesh structure, of about 30 μm.

Comparative Example 1

The present comparative example relates to a microfluidic device whichuses the surface of a known amino group-fixing glass substrate for thebottom surface of a channel, and indicates that the DNA fixation densityis low.

[Formation of Indentation (Channel)]

Composition (X1) produced in Example 1 was coated at a rotating speed of800 rpm using a spin coater (Mikasa) onto a slide glass in which aminogroups were introduced onto the surface (Amine Silane, Matsunami GlassIndustries) to form an uncured coating of said composition (X1), saiduncured coating was irradiated for 120 seconds with ultraviolet lightfrom ultraviolet lamp 2 through a photomask at locations other thanwhere the channel is to be formed to form a semi-cured coating of saidcomposition (X1), and the non-irradiated portion of uncured composition(X1) was removed with ethanol to form an indentation (channel 4) wherethe glass was exposed on the bottom surface.

[DNA Fixation]

The aforementioned indentation (channel 4) having amino groups on itsbottom surface was subjected to glutaraldehyde treatment using the samemethod as Example 1 to introduce aldehyde groups onto the bottomsurface.

Next, the aforementioned indentation (channel 4) in which aldehydegroups were introduced was treated in the same manner as Example 1 tocarry out DNA fixation.

[DNA Quantification]

As a result of measuring the aforementioned indentation (channel 4) inwhich DNA was fixed to glass on the bottom surface in the same manner asExample 1, the value of fluorescent intensity was 73 LAU/mm².

Comparative Example 2

The present comparative example indicates that the amount of DNA fixedto a silicon substrate surface is low. However, measurements wereperformed without forming an indentation.

[Introduction of Amino Groups]

After pre-treating a silicon wafer by irradiating with ultraviolet lightin a vacuum (Sen Koki), the wafer was immersed in a 1 mM isopropanolsolution containing a silane coupling agent having amino groups in theform of 3-aminopropyl triethoxysilane (LS-3150, Shin-Etsu Silicones) for3 hours at 25° C. followed by washing with ethanol and hot air dryingfor 30 minutes at 80° C. to introduce amino groups onto the surface ofthe silicon wafer.

[DNA Fixation]

Glutaraldehyde treatment and amino group modification DNA treatment werecarried out in the same manner as Example 1 on the surface of theaforementioned silicon wafer on which amino groups had been introducedto fix DNA to the surface of the silicon wafer.

[DNA Quantification]

As a result of measuring the surface of the aforementioned silicon waferon which DNA had been fixed using the same method as Example 1, thevalue of fluorescent intensity was about 78 LAU/mm².

Comparative Example 3

The present comparative example indicates that the amount of DNA fixedto a silicon porous layer is low. However, measurements were performedwithout forming a channel.

[Formation of Porous Channel]

A porous layer having a width of 250 μm and a depth from the surface ofa silicon wafer of about 30 μm was formed in accordance with the processdescribed in Japanese Unexamined Patent Application, First PublicationNo. H6-169756 using a silicon wafer.

[Introduction of Amino Groups]

Amino groups were introduced into this porous layer in the same manneras Comparative Example 2.

[DNA Fixation]

DNA was fixed to this porous layer in the same manner as ComparativeExample 2.

[DNA Quantification]

As a result of measuring the surface of the aforementioned porous layeron which DNA had been fixed to bottom surface porous silicon using thesame method as Example 1, the value of fluorescent intensity was about114 LAU/mm². Namely, fluorescent intensity only increased about 1.46times as compared with a smooth silicon wafer surface.

Comparative Example 4

The present comparative example indicates that the amount of DNA fixedto surface having surface irregularities formed by electron beam etchingis low.

[Formation of Surface Irregularities]

An acrylic sheet was etched with an electron beam for 5 minutes at 15 mAusing the Model E1030 Ion Sputtering System (Hitachi) to provide surfaceirregularities having a depth of about 0.4 μm in the surface.

[Production of Microfluidic Device]

A microfluidic device was produced by carrying out epoxy groupintroduction, channel formation, amino group introduction, aldehydegroup introduction, DNA fixation and cover adhesion in the same manneras Example 2 with the exception of using an acrylic sheet in whichsurface irregularities were formed in the manner described above insteadof an acrylic sheet on which was formed a porous resin layer having athree-dimensional mesh structure.

[DNA Quantification]

As a result of measuring in the same manner as Example 2, the value thevalue of fluorescent intensity was about 288 LAU/mm².

According to the DNA quantification results of the aforementionedExamples 1 to 3 and Comparative Examples 1 to 4, in comparison with thechannel not having a porous resin layer of Comparative Example 1, achannel having a porous bottom surface made of silicon, or a channelhaving a simple irregular surface instead of a porous channel having athree-dimensional mesh structure, the specific surface area of thechannels provided with a porous resin layer having a three-dimensionalmesh structure of the examples was extremely large, and were clearlyable to fix numerous substances.

INDUSTRIAL APPLICABILITY

Since a microfluidic device of the present invention has a thin porousresin layer adhered to the inner surface of a channel, the fixation oflarge amounts of functional groups, (bio)chemical substances orbiological substances can be realized even in a minute region withoutobstructing the channel. In addition, since said porous resin layer hasa three-dimensional mesh structure, said porous resin layer has anextremely large surface area, enabling it to fix a large amount of aprobe. Moreover, a microfluidic device provided with a porous resinlayer having a three-dimensional mesh structure on one or two innersurfaces of the aforementioned rectangular or trapezoidal channel, andparticularly a microfluidic device in which the average distance fromsaid porous resin layer to an opposing inner wall is within the range of1 to 50 μm, is able to generate effective affinity between an analysistarget substance that moves over said porous section and a fixed probe.Consequently, when a microfluidic device of the present invention isused, treatment such as synthesis, separation or analysis can be carriedout accurately and quickly. In particular, a microfluidic device of thepresent invention can be preferably used for DNA analysis by using anoligonucleotide for the fixed probe.

1. A microfluidic device comprising a substrate, a porous resin layerhaving a three-dimensional mesh structure, a channel and a cover;wherein, said microfluidic device (I) has said porous resin layer in theupper portion of the substrate, (II) said porous resin layer is filledwith a curable resin of an activating energy beam-curable resincomposition (X) impregnated excluding the channel portion, and (III) thechannel has wall surfaces consisting of a porous resin layer having athree-dimensional mesh structure that is not filled with activatingenergy beam-curable resin composition (X), a curable resin layer of theactivating energy beam-curable resin composition (X) formed in the upperportion of the porous resin layer having a three-dimensional meshstructure filled with the curable resin of activating energybeam-curable resin composition (X), and a cover, and is formed into theform of a cavity.
 2. The microfluidic device according to claim 1,wherein the porous resin layer having a three-dimensional mesh structureis composed of an activating energy beam-curable resin composition. 3.The microfluidic device according to claim 1, wherein, in across-section in the direction perpendicular to the direction of flow ofa fluid in a portion of the channel having a porous resin layer having athree-dimensional mesh structure, if an arbitrary point in saidcross-section is designated as x, the portion of the porous resin layerhaving a three-dimensional mesh structure that is closest to saidarbitrary point in terms of straight line distance is designated as y,the straight line distance between x and y is designated as r, and themaximum distance that r is able to have in said cross-section isdesignated as r^(max), then said r^(max) is within the range of 1 to 50μm.
 4. The microfluidic device according to claim 1, wherein the shapeof a cross-section of the channel in the direction perpendicular to thedirection of fluid flow is rectangular or trapezoidal.
 5. Themicrofluidic device according to claim 4, wherein the porous resin layerhaving a three-dimensional mesh structure is formed on only one innerwall of the channel, and the average distance from said porous resinlayer to the opposing inner wall is within the range of 1 to 50 μm. 6.The microfluidic device according to claim 1, wherein the thickness ofthe porous resin layer having a three-dimensional mesh structure iswithin the range of 0.5 to 30 μm.
 7. The microfluidic device accordingto claim 1, wherein the mean pore diameter of the porous resin layerhaving a three-dimensional mesh structure is within the range of 0.05 to3 μm.
 8. The microfluidic device according to claim 1, wherein theporous resin layer having a three-dimensional mesh structure is a porousresin layer to which a probe having affinity for an analysis targetsubstance is fixed.
 9. The microfluidic device according to claim 8,wherein the probe is an oligonucleotide.